Detection and modification of gut microbial population

ABSTRACT

Methods and systems for evaluating heath/disease state of an individual are provided herein. In particular, the disclosure provides methods for measuring levels of one or more metabolites of interest in an individual and using these measurements to assess the individual&#39;s health. The disclosure also provides systems for carrying out the disclosed methods. For example, the disclosed systems may include a metabolite level evaluation module to determine the individual&#39;s heath based on the metabolite levels measured.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.16/859,453, filed Apr. 27, 2020, now U.S. Pat. No. 11,041,847, which inturn is a continuation-in-part (CIP) application of U.S. applicationSer. No. 16/258,199, filed Jan. 25, 2019, now abandoned, the contents ofwhich are incorporated herein by reference.

FIELD

The present disclosure relates generally to systems and methods forevaluating health or disease state of an individual, and for improvingthe health of the individual. In particular, the disclosed systems andmethods measure the levels of one or more metabolites of interest in theindividual to determine a current state of the gut microbial populationof an individual, and provide a personalized program for improving thehealth of the individual. Specifically, the present disclosureidentifies and describes techniques for measuring levels of certain keymetabolites, and using the measured metabolite levels to assess heathstate/disease state or disease risk of the individual. The presentdisclosure also describes methods for providing behavior modification,e.g., relating to nutrition, physical training, sleep, or mentalwellness, and for providing supplements, to improve health, diseasestate, or disease risk of an individual, based on the measuredmetabolite levels of the individual.

SUMMARY

The gut is colonized by many (6-10,000) different species of bacteria,viruses, parasites, fungus and archaea. Diversity of the gut microbiomeis dependent on many factors, e.g., genetics, illness, diet, and druguse, and thus there is large individual variation of the population inthe gut. At times, there exists a need to evaluate an individual'smicrobiome. Many microbiome tests employ stool samples and only measurecertain species of bacteria or other microorganisms present in the stoolsample. While stool sample tests often can identify the presence of somebacteria, stool sample tests are difficult to collect and only revealthe existence of a limited number and species of bacteria, etc., presentin the gut. As such, stool sample tests cannot provide guidanceregarding overall health, disease state or disease risk of anindividual, or recommend actions for improving health disease state ordisease risk.

Other factors also must be considered. First all of the species in thegut are essentially in a Darwinian competition for survival dominanceand activity. This essentially means that the mere existence of aspecies in the stool does not necessarily indicate its activity ineither utilizing or producing compound that leaves the gut to carry outfunctions in the rest of the body. Second the functionality of the gutmicrobiome is affected not only by environmental behavioral factors(diet, stress, exercise etc.) but by the underlying genome of theindividual. As will be discussed below, we have shown this in studies ofthe small molecule footprint in feces by which young just weaned genemodified and wild type littermate mice could be uniquely discriminatedfrom each other. Same cage-same mother-same microbiome-sameenvironmental exposures different gut functionality.

Thus measuring the result of microbiome activity as it affects levels ofmicrobiome modified compounds in the plasma, whole blood, or DBS allowsdirect understanding of the role of the microbiome. This provides a setof essential materials for monitoring and optimizing health and riskthrough control/modification of the microbiome functionality.Embodiments of the present disclosure provide methods and systems forindirect measuring levels of certain key metabolites of the gutmicrobiome that effect system health of an individual and take intoaccount the effect of different microbial species as well as theimportant metabolic pathways influenced by the gut microbiome, bymeasuring certain microbiome modified compounds in a blood sample of anindividual. More particularly, we have found that certain keymetabolites of the gut microbiome which can be indirectly measuredthrough the presence of certain microbiome modified compounds in a bloodsample, play an outsized role in effecting many aspects of health of anindividual, including gut health, emotional balance, cognitive acuity,energetic efficiency and immuno fitness, and can easily be adjusted toimprove the health state of the individual. These particular keymetabolites are indole propionic acid (IPA), indole-3-lactic acid (ILA),indole-3-acetic acid (IAA), Tryptophan (TRP), Serotonin (SER),Kynurenine (KYN), Total Indoxyl Sulfate (IDS), Tyrosine (TYR), Xanthine(XAN), 3-Methylxanthine (3MXAN), and Uric Acid (UA).

In one aspect the present disclosure provides a method for improving thehealth or disease state of an individual, by indirectly measuring theconcentration of certain key metabolites of the gut comprisingcollecting liquid samples from the body of said individual and analyzingsaid liquid samples for concentration of two or more of the followingmicrobiome modified compounds selected from the group consisting of:indole propionic acid (IPA), indole-3-lactic acid (ILA), indole-3-aceticacid (IAA), Tryptophan (TRP), Serotonin (SER), Kynurenine (KYN), TotalIndoxyl Sulfate (IDS), Tyrosine (TYR), Xanthine (XAN), 3-Methylxanthine(3MXAN), and Uric Acid (UA); comparing the concentration of saidselected microbiome modified compounds to standards; and, prescribing atreatment protocol to raise or reduce the concentration, as the case maybe, for those metabolites that fall outside of an accepted range.

In one embodiment, the liquid sample preferably comprises blood,although urine, saliva or tears also could be tested.

In another embodiment, a minimum of three, four, five, six, seven,eight, nine, ten, eleven or twelve of said metabolites are analyzed.

In another embodiment, the treatment protocol includes administration ofone or more supplements selected from the group consisting of aprebiotic, a probiotic, melatonin, a vitamin B complex, copper, andCoQ10.

The microbiome modified compounds are described below.

Indole Propionic Acid (IPA) is produced in the human gut exclusively byone species of gut bacteria (Clostridium sporogenes) from tryptophan.Research indicates that if dysbiosis is occurring, these are amongst thefirst bacteria to be negatively impacted, resulting in lower IPA levels.Thus, IPA is a critical marker for a healthy gut microbiome. IPA is alsoconsidered to be one of the strongest antioxidants in the body.Oxidative stress is a damaging process that results in tissue damage andcell death and IPA has been shown to protect cells from oxidative stressdamage, which plays an important role in a variety of systems healthincluding brain health. Additionally. IPA plays a crucial role inmaintaining a healthy gut by strengthening the tight junctions (spaces)between the cells lining the gut. This is important for maintainingintestinal structural integrity and decreasing intestinal permeability.Intestinal permeability can lead to substances exiting the intestineinto the bloodstream, leading to conditions such as food allergies.

Indole-3-Lactic Acid (ILA) is a tryptophan metabolite and precursor toindole-3-propionic acid (IPA) and is important for strong internalfitness. Low levels of ILA may prevent the formation of adequate amountsof downstream metabolites such as indole-3-propionic acid (IPA). Lowlevels of ILA may also indicate low levels of Bifidobacterium in theintestine. Bifidobacterium can assist with conversion of tryptophan toILA and low levels of Bifidobacterium can lead to further dysbiosis.However, high levels of ILA may also lead to gut dysfunction anddiscomfort because, at high levels, ILA can be toxic to the beneficialbacteria in the microbiome. Excessive high levels of ILA may alsoindicate dysbiosis of the gut metabolome, because the bacteriumClostridium sporogenes that uses ILA to make indole-3-propionic acid(IPA) may be too low to effectively convert ILA to IPA. If the quantityof beneficial bacteria decreases significantly, there is a chance thatinvasive bacterial species can proliferate, leading to furtherdysbiosis.

Indole-3-Acetic Acid (IAA) is an indirect precursor to IPA and is alsoan auxin produced in plants. IAA has been shown to possess antioxidantactivity and low levels of indole-3-acetic acid (IAA) are generally nota problem. However, low IAA should be considered in the context of othermetabolites in the tryptophan branch, specifically indole-3-propionicacid (IPA). If both IAA and IPA are low, this can be a marker of gutdysbiosis. High levels of IAA have been reported to inhibit the growthand survival of the beneficial Lactobacillus species of gut bacteria,which converts sugars such as glucose and fructose to lactic acid, whichmay inhibit the growth of some harmful bacteria. High levels of IAA alsohave been associates with poor kidney and heart health.

Tryptophan (TRP) is obtained mainly through one's diet. Certain gutbacteria are also capable of making tryptophan. Tryptophan is an aminoacid. Amino acids are building blocks for proteins that are essentialfor maintaining a healthy body. Some of these proteins includeantibodies (for immunity), hormones (for signaling), muscles (formovement), and enzymes (for biochemical reactions). Tryptophan can crossthe blood-brain barrier, making it available to produce importantneurotransmitters like serotonin, which is essential for normal brainprocesses that affect mood, behavior, memory, and learning. Manydigestive disorders can change tryptophan levels. Factors like stress,gut dysbiosis, and infection enhance tryptophan degradation, resultingin lower tryptophan levels. Low tryptophan levels have been associatedwith gut dysbiosis and “fad dieting” and have also been linked tooccasional difficulties sleeping and muscle aches and pains. High levelsof tryptophan may also be the result of gut dysbiosis. In some suchcases, the gut microbiome may not be breaking down the tryptophanefficiently to create other necessary metabolites. Higher levels oftryptophan have been observed after major surgeries and in subjects withhigh protein diets. High levels of tryptophan have been linked to moodswings, increased aggressiveness, and tremors.

Serotonin (SER) is a tryptophan metabolite. Serotonin synthesis isfacilitated by the adequate presence of vitamins B1, B3, B6, and folicacid. Serotonin is one of the most important signaling molecules withinthe gut, where it plays a pivotal role in initiating secretions(mucosal) and motor reflexes (the movement of the intestine) and iscrucial for normal functionality of the central nervous system. Most ofthe body's serotonin is produced by cells in the intestinal wall.Approximately 95% of circulating serotonin originates from these cells.Gut dysbiosis or an inflammation of the gut may interfere with theenterochromaffin cells that make serotonin in the intestinal lining. Lowlevels of serotonin have been linked to occasional sleep problems,muscle aches and pains, poor brain health, and poor gut health. Highlevels of serotonin can be linked to shivering, diarrhea, and muscletightness. Additionally, high levels of serotonin have also been linkedto occasional agitation, restlessness, confusion, poor heart health,dilated pupils, muscle aches and pains, sweating, headaches, and goosebumps.

Kynurenine (KYN) is a metabolite of tryptophan. Its primary function isto dilate blood vessels during an inflammatory response. Kynurenine isalso a regulator of the immune system. Kynurenine is used in theproduction of niacin (vitamin B3). Niacin is considered to be one of theessential human nutrients as it participates in DNA repair. Kynurenineis utilized by the endocrine system to produce certain hormones.Kynurenic acid, a metabolite of kynurenine, is a neuroprotective agentin the brain. Its production is mediated by vitamin B6, selenium, andsulfur containing amino acids. Kynurenine also acts in the eye toprotect against UV radiation. Low levels of kynurenine may be caused bygut imbalances and low levels of kynurenine can lead to low levels ofdownstream metabolites (kynurenic acid) and vitamin B3. Low levels ofkynurenine have also been linked to decreased energy and gut dysbiosis.The most common cause of elevated kynurenine and its metabolites is theexcessive use of tryptophan supplementation. Additional causes includethe presence of chronic infections and vitamin B6 deficiency. Changes inthe ratio of kynurenine to tryptophan are an indication of a generalhealth imbalance. Exercise can be a key modulator of kynurenine becauseit reduces kynurenine levels and thus reduces excretion of itsdownstream metabolic product, kynurenic acid. Total Indoxyl Sulfate(IDS) is a metabolite of tryptophan and is absorbed into the blood fromthe liver. IDS is a toxin made from a gut bacteria metabolite in theliver and is a molecule known as a uremic toxin. High levels of IDS hasbeen associated with poor kidney and heart health. Low levels of indoxysulfate (IDS) are considered positive. Research suggests that normallevels of indoxyl sulfate may act as an antioxidant and high levels ofIDS can lead to oxidative stress in numerous cell types, includingvascular smooth muscle cells, endothelial cells, and bone cells. Highlevels have also been found in people who take too much tryptophansupplement.

Tyrosine (TYR) is a nonessential amino acid, which means that anindividual can synthesize it from another amino acid, phenylalanine.Tyrosine is present in almost every protein in the body. Tyrosinefunctions as a building block for several important neurotransmitters,such as dopamine, epinephrine, and norepinephrine. Neurotransmittersregulate mood, behavior, and general feelings of well-being. Tyrosine isneeded for the thyroid gland to produce hormones T3 and T4 (whichregulate growth, metabolism, body temperature, and heart rate). Tyrosineis also an important amino acid for the pituitary gland. The pituitarygland, like the thyroid gland, controls metabolism and growth;additionally, it regulates sexual maturation, reproduction, bloodpressure, and many other vital physical processes. Tyrosine is alsonecessary for cell division. Low levels of tyrosine are rare; however,some researchers believe that stress may affect the body's ability tomake tyrosine. Low levels of tyrosine can lead to poor brain health andabnormally high levels of tyrosine can lead to poor gut and brain healthand have also been linked to anxiety. High protein diets can raisetyrosine levels. High levels of Tyrosine may also be the result of a gutdysbiosis when, for example, the body's gut microbiome is not breakingdown the tyrosine efficiently into other metabolites. High levels oftyrosine are also associated with a genetic disorder called tyrosinemia.This condition is caused by the deficiency of one of the enzymesrequired for the multistep process that breaks down tyrosine.

Xanthine (XAN) is a metabolite of the purine pathway and functions inthe digestive tract to induce hydrochloric acid production and promotesecretion of pepsin from cells lining the stomach. Both of theseprocesses help to break down consumed food. Xanthine is also a mildstimulant and is found in coffee, cola, and green tea. Very low levelsof xanthine are rarely seen except in cases of a genetic disorder calledxanthinuria. Low levels of xanthine can lead to digestive discomfort dueto the role xanthine plays in inducing digestive enzymes. Xanthinelevels at the low end of normal can be associated with oxidative stress.High levels of xanthine can be caused by overconsumption of caffeinatedbeverages and by certain medications (bronchodilators), as well as highpurine diets. High levels of xanthine have been linked to nausea,elevated heart rate, and poor heart health. 3-Methylxanthine (3MXAN) isa purine metabolism-breakdown product in caffeine and theophylline.Metabolites of the purine pathway are significant because they affectboth the gut and the brain. In the gut, 3MXAN increases hydrochloricacid and pepsin secretion, which aid in digestion. Purines are keycomponents of cellular energy systems (e.g., ATP and NAD), cellularsignaling, and along with pyrimidines are involved in RNA and DNAproduction. Presently there is limited research to indicatephysiological effects when low levels of 3-methylxanthine (3MXAN) arepresent. However, because methylxanthines increase hydrochloric acid andpepsin secretion in the GI tract, slow digestion might be aided byconsuming a cup of tea with or after a meal. High levels of 3MXAN can bean indication of medication used for chronic obstructive pulmonarydisease (COPD) and asthma or of excessive intake of caffeine products.Individuals suffering from acid reflux are encouraged to limit theirintake of methylxanthines. It is thought that ahigh-carbohydrate/low-protein diet can decrease the removal of 3MXANfrom the blood at a normal rate, accordingly, a diet high in fiber andprotein may reduce 3MXAN levels.

Uric Acid (UA) is the final product of human purine metabolism. Uricacid may be a marker of oxidative stress. Approximately ⅓ of purines arederived from diet. The majority of uric acid is dissolved in the blood,filtered through the kidneys, and expelled in the urine. Low levels ofuric acid may be an indication of over hydration or a low-purine diet.High levels of uric acid can be hereditary or could be a symptom ofgout. High levels can also be caused by temporary dehydration, excessivebeer drinking, and/or a high-purine diet. It has been reported thatfructose intolerance can be indicated by high levels of uric acid.

4-Hydroxybenzoic Acid (4HBAC) is believed to also be a key metabolite ofthe gut microbiome. However, 4HBAC is somewhat unstable and cannot bereliably measured. Notwithstanding, 4HBAC also is included as ametabolite of interest, as discussed below. 4HBAC is found naturally inlentils, vanilla, gooseberries, and mushrooms. 4-Hydroxybenzoic Acid hasalso been found in humans after drinking green tea and wine. 4HBAC is apopular antioxidant because of its low toxicity. 4HBAC and its precursorbenzoic acid are produced by bacteria in the gut and can be used as anindicator of intestinal dysbiosis or leaky gut syndrome. Esters of 4HBACare referred to as parabens and are used widely as antimicrobialpreservatives in cosmetics, for pharmaceutical applications, and inprocessed foods. Low levels of 4-hydroxybenzoic acid (4HBAC) do notappear to have any adverse physiological effects; however, low levelsmay be an indication that ingested natural antioxidants are in lowquantity. Abnormally high levels of 4HBAC can lead to intestinaldysbiosis and can also be an indication of increased intestinalpermeability (“leaky gut”). Exposure to high concentrations of processedfoods or skin creams with parabens can increase levels of 4HBAC.Parabens functionally mimic estrogens and have been associated with lowenergy and poor skin health. Overindulgence in fruits such asblueberries can increase the levels of 4HBAC.

In practice for dietary recommendations these micrbobiome modifiedmolecules are not considered singly but as an entire network. In thesimplest form this is as ratios of each compound to all other compounds(I. E. 78 separate variables). As an example KYN to TRP is a ratio thatin the normal population has a relative standard deviation ofapproximately 30% with a within individual variation from severalsamples of approximately 10%. However, concussion will elevate thisratio as well certain types of cancers and other diseases. Normal levelsare related to gut functionality. Similarly low IPA, high UA, and IAAindividually constitute risk factors for cardiovascular disease.Considered together they modify the assessment of risk in evaluation ofmediation strategies. In a more complex data treatment, given a largernumber of samples and outcomes, the data can be organized using groupingof profiles versus outcome techniques such as PCA (unsupervisedprincipal component analysis) or PLSDA (partial least squaresdiscriminate analysis) using Umetrics Simca-P software available fromSartorius Stedim Biotech and Matlab software available from MathWorks,Inc., or correlation networks could be used as taught in prior U.S. Pat.No. 6,194,217, the context of which are incorporated herein byreference.

In contrast with previous techniques of evaluating the gut microbiomethrough stool sampling, the presently disclosed systems and methods areable to indirectly measure key metabolites of the gut by measuring thefunctional effect of how the gut microbiome is working (the output ofthe organisms in the gut) and how these organisms interact with the gutto put necessary nutrients and occasionally undesirable compounds intothe blood. Only knowing what is in the gut microbiome does not provideinformation regarding how the gut is functioning to provide biochemicalsthat run and control the body. Thus, a stool sample alone provides anincomplete picture of an individual's health/disease state.

Molecules called short chain fatty acids (SCFAs) are created by bacteriain the gut during the fermentation process from carbohydrates andproteins. SCFAs have several effects in the body including epithelialcell transport and metabolism. SCFAs also provide energy sources formuscles and kidneys, as well as for the heart and brain. Because 95% ofmetabolites created by SCFAs are absorbed from the gut, fecal analysisof SCFAs and their metabolites is not particularly useful in healthypeople. On the other hand, being able to measure the “output” of SCFAscan provide insight into whether the gut is functioning properly.

In some aspects, the disclosed techniques allow correlations betweenvarious functions of the body to be observed (for example, correlationsbetween exercise and the gut microbiome). The disclosed systems andmethods may also, in some embodiments, measure the output of bacteriapresent in the microbiome, which indicates whether specific compounds inthe body are where they need to be and may allow an individual to usethis information to make alterations to improve their metabolite levels.

In various embodiments, a blood sample from an individual is used toevaluate output from bacteria in the microbiome. It has been found thatblood samples can capture a significant part of the complexity ofmicrobiome interactions. The results from measuring some criticalnutritional, energetic, control, and protective markers present in ablood sample can be used to modify and monitor parameters that reflectsuboptimal function. Based on the profile of the metabolites measured ina blood sample, one or more recommendations can be made from a detailedmeta-analysis of available peer reviewed literature, internal data andstudies, and general best practice in nutrition, training, andsupplementation. The features, functions, and advantages that have beendiscussed can be achieved independently in various embodiments of thepresent disclosure or may be combined in yet other embodiments, furtherdetails of which can be seen with reference to the following descriptionand drawings. Other features, functions and advantages of the presentdisclosure will be or become apparent to one with skill in the art uponexamination of the following drawings and detailed description. It isintended that all such additional systems, methods, features, andadvantages be included within this description, be within the scope ofthe present disclosure, and be protected by the accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Many aspects of the disclosure can be better understood with referenceto the following drawings. The components in the drawings are notnecessarily to scale, emphasis instead being placed upon clearlyillustrating the principles of the present disclosure. Moreover, in thedrawings, like reference numerals designate corresponding partsthroughout the several views.

FIG. 1 shows an exemplary system for selective metabolite measurementand analysis, in accordance with some embodiments of the subjectdisclosure.

FIG. 2 shows a flow diagram of an exemplary method for testing andanalyzing metabolites of interest, in accordance with some embodimentsof the subject disclosure.

FIGS. 3A-3D show scored test reports of microbiome health of anindividual in accordance with the subject disclosure.

FIG. 4 is a three-dimensional graph of patterns of biochemicalmeasurements of ALS Mouse and Wild type littermate and treated micesamples.

FIGS. 5A and 5B are graphs of IPA measurements of wild type and genepositive HD mouse models.

DETAILED DESCRIPTION

In the following description, reference is made to the accompanyingdrawings, which form a part hereof, and in which is shown, by way ofillustration, various embodiments of the present disclosure. It isunderstood that other embodiments may be utilized and changes may bemade without departing from the scope of the present disclosure.

Theory

After significant research, the inventors of the subject disclosure,without being bound by theory, have determined that the body is believedto operate as a type of “Omic web” in which interactions of the genome,transcriptome, proteome, metabolome, and microbiome are linked andaffect one another. It therefore follows that the health/disease stateor disease risk of one system can be assessed by measuring certaincomponents of a different system (e.g., the state of the genome can beassessed by measuring components of the microbiome). Extensive research,evaluation, and experimentation has revealed that certain metabolites inan individual appear to reliably and accurately determine thehealth/disease state or disease risk of an individual. In other words,the Omic web of an individual can be well, if not completely, describedby measuring the certain metabolites of interest. Furthermore, uponassessment of these metabolites, tailored recommendations can beprepared to improve the health or disease risk of the individual.

As discussed in detail below, the disclosed systems and methods in oneaspect involve the measurement and analysis of certain key metabolitesof interest, in particular, indole propionic acid (IPA), indole-3-lacticacid (ILA), indole-3-acetic acid (IAA), Tryptophan (TRP), Serotonin(SER), Kynurenine (KYN), Total Indoxyl Sulfate (IDS), Tyrosine (TYR),Xanthine (XAN), 3-Methylxanthine (3MXAN) and Uric Acid (UA).4-Hydroxybenzoic acid (4HBAC), also is believed to be a key metaboliteof the gut microbiome metabolites; however, 4HBAC is somewhat unstable,and thus cannot be reliably measured. Notwithstanding, analyzing for4HBAC can providing useful information in certain situations.

In another aspect, the disclosed system and methods involve modificationand monitoring of the gut microbiome for treating deficiencies of thegut microbiome.

Example Systems and Methods

Systems and methods for measuring metabolites of interest and using themeasured metabolites to evaluate an individual's health or disease stateor disease risk are described herein. As used herein, the terms “healthstate”, “disease state” and “disease risk” refer to the overall physicalhealth of an individual, including an individual's risk for developingone or more diseases or health conditions. FIG. 1 shows an exemplarysystem 100 for carrying out the disclosed methods. FIG. 2 illustrates anexemplary method 200 for measuring metabolites of interest andevaluating the measured metabolite levels to assess an individual'shealth or disease state or disease risk. As used herein, the term“individual” means any human or non-human mammal. As shown in FIG. 1,system 100 includes a sampling device 102, which may be used to obtain asample from an individual. Sampling device 102 may be designed toaccommodate any type of sample of bodily fluid from an individual, suchas a blood sample, although a urine sample and/or a saliva sample and/ora sweat sample also advantageously may be used.

In select embodiments, sampling device 102 is configured to receive awet blood sample (e.g., with one or more layers of filter paper). Insome such embodiments, the blood sample may be obtained from theindividual by any method known in the art. This may include traditionalvenipuncture, wherein blood is obtained directly from an individual'svein. The blood may also be collected via a finger prick or via a“prick” of any other part of the individual's body. Blood collected viathis method is typically obtained from blood capillaries near thesurface of the skin by piercing the skin with a lancet or similardevice. The blood from the “finger prick” may be collected into acapillary tube and then dispensed onto a substrate of sampling device102. Any desired sample volume may be used, such as, for example, lessthan 10 mL, less than 5 mL, or, in some cases less than 1 mL. In someembodiments, sampling device 102 may allow a wet blood sample topartially or fully dry to produce a dried sample 104. In a preferredembodiment the sampling device comprises a blood sampling device asdescribed in US 2016/0320367, the contents of which are incorporatedherein by reference.

Method 200 continues with solubilizing 202 the dried sample 104 to yielda solubilized sample 106. Dried sample 104 may be solubilized 202 withany desired solvent or solvent mixture. In some embodiments, forexample, deionized water, and/or other suitable solvents such asacidified acetonitrile (0.4% acetic acid in acetonitrile), may be usedto solubilize 202 dried sample 104. During solubilization 202,metabolites present in dried sample 104 are transferred to solubilizedsample 106. In some embodiments, solubilized sample 106 may be separatefrom sampling device 102 while, in other embodiments, solubilized sample106 may be contained inside sampling device 102.

Method 200 continues with optionally processing 204 the solubilizedsample 106. In some embodiments, processing 204 solubilized sample 106may include heating, cooling, diluting, concentrating, separating,adjusting pH, and/or introducing one or more binding agents tofacilitate metabolite measurement.

Method 200 continues with measuring 206 one or more of the keymetabolites of interest present in solubilized sample 106. Any suitablenumber of metabolites of interest may be measured 206 in the disclosedmethods and systems. For example, in some embodiments, several,preferably eleven or all twelve of the above mentioned key metabolitesof interest may be measured. In some particular embodiments, between aminimum of two, and preferably between three and seven key metabolitesof interest may be measured. In other select embodiments, 8, 9, 10, 11,or 12 of the key metabolites of interest may be measured. The keymetabolites of interest are selected based on their outsized role ineffecting many aspects of health, as well as their “actionability,”i.e., whether action can be taken to positively modify the level ofmetabolite present.

The selected key metabolites of interest may be measured 206 accordingto any known technique, including with a metabolite measurement device108 (as illustrated in FIG. 1). Metabolite measurement device 108 mayrely on analytical chemistry techniques, such as a two column liquidchromatography (LC), followed by a 16 channel electrochemical arraydetector, following the teachings of my prior PCT/US98/22275 and usingas an A phase solvent 0.1MLi₃PO₄ and 0.5% Methanol, and as a B phasesolvent 0.1MLi₃PO₄ and 35% Acetonitrile. The levels of the selectedmetabolites of interest measured in an individual's blood sample arecompiled in a chart, graph, list, or other form, and compared tostandards as described below. FIG. 1 includes a chart 110 displayingmeasured key metabolites of interest. In select embodiments, measuredlevels of key metabolites of interest may be provided inmachine-readable format. A summary of the measured key metabolites ofinterest may be provided in a format designed for a medical professionalor for an individual, depending on intended application.

Method 200 continues with determining 208 heath and/or disease statebased on the measured key metabolites of interest. In some embodiments,health and/or disease state of an individual can be determined using ametabolite level evaluation module 112, as shown in FIG. 1. Metabolitelevel evaluation module 112 may be implemented on a processor or othertype of computing device. Metabolite level evaluation module 112 may, insome embodiments, be configured to receive levels of measured keymetabolites of interest as an input, evaluate the measured levels ofmetabolite of interest, and output an analysis of health/disease statebased on peer-reviewed literature which was validated and confirmedinternal studies of hundreds of individuals based on their extensivehealth histories, and configured to calculate health category scorescalculated by “weighing” certain metabolites based on their importancein that category.

In some embodiments, metabolite level evaluation module 112 may beconfigured to output health/disease state results 114, as shown inFIG. 1. Health/disease state and disease risk results 114 may include,in some cases, a report, a listing of the levels of the key metabolitesof interest measured, and/or recommendations for modifying the gutmicrobiome in order to improve the health/disease state or disease riskof the individual. In particular embodiments, health/disease or diseaserisk state results 114 include an evaluation of one or more of thefollowing: a summary of gut health, immune-fitness, cognitive acuity,emotional balance, and energetic efficiency.

Method 200 concludes with optionally preparing recommendations 210 basedon the health/disease state or disease risk of the individual. Forexample, if desired, the compiled health/disease state or disease riskresults 114 may include recommended actions for health improvement 116,as shown in FIG. 1. Example recommended actions for health improvement116 include nutritional changes, more particularly ingestion of tailoredsupplements, and optionally also may include one or more of thefollowing types of recommendations: physical training, sleep schedule,emotional balancing techniques (e.g., therapy, medication, etc.), andlifestyle (e.g., interventions aimed at reducing stress, improving sleephygiene, and/or mindfulness training). The compounds measured arerelated in a web of interaction among themselves and among differentorgans, bacteria composition and environmental factors (diet, exercise,stress and medications) and the individual's underlying genetic makeup.The tests and treatment recommendations can beneficially modifyeverything but the final factor. In select embodiments, nutritionalrecommendations may relate to food groups specific to each keymetabolite, feeding plans specific to energy requirements, and/oreducational information regarding ways to improve the levels of keymetabolites of interest present in the individual's body.

The output of the several key metabolites are then scored and compiledinto the history chord of microbiome health for the tested individual,(FIGS. 3A-3D), and based on the scoring and individual concentrations ofthe several key metabolites as above discussed, a program including dietchange, exercise and lifestyle habit changes be proposed together withtailored supplements chosen specifically to modify levels of the keymetabolites, as needed. Also, a schedule is created to monitor changesin key metabolites by periodically testing and adjustment of tailoredsupplements until an optimal microbiome health score is achieved for theindividual. Supplements include prebiotics, probiotics, viablemelatonin, vitamin B complex, copper, and CoQ10 as will be discussedbelow.

Nutritional change recommendations include supplement recommendations,as follows:

-   1. (Probiotic)—Helps to support the balance the good bacteria in the    gut. Biome support contains 17 species of bacteria. It is designed    to help normalize interactions between the multiple species in the    gut. The use of biome support is suggested when biomarkers such as    IPA, IIA, IAA and IDS are found to be outside of normal, reported    and cited concentrations. The use of a probiotic as a general    supplement is confirmed typically by a serial testing after 2 to 3    and 6 months of use.-   2. (Tryptophan)—Helps support neurotransmitter production, vitamin    production and sleep. Tryptophan is only suggested to be used when    dietary interventions prove to be inadequate at maintaining the    appropriate relationship between the ratios of Tryptophan to    Tyrosine, due to the fact that Tryptophan and Tyrosine share the    same LAT1 transporter across the blood brain barrier with Branch    Chain Amino Acids (BCAA) which are frequently used by athletes. This    individual factor needs to be considered in the use of Tryptophan    (also a precursor to sleep regulating hormones). Tryptophan is    advantageously used for improving Energetic Fitness, Cognitive    Acuity, Emotional Balance, Gastro Intestinal Fitness, Immuno Fitness    as discussed below.-   3. (5HTP and B6)— Helps support the production of neurotransmitters,    gut mobility, and sleep. 5HTP and B6 are recommended when both    Tryptophan and serotonin is low and certain prescription drugs are    not being taken. 5HTP is the precursor for Serotonin. In situations    sleep difficulties are reported problem by tryptophan is in a normal    range Balance might be recommended. In the gut Tryptophan is    frequently n-acetylated. N-acetyl tryptophan can proceed more easily    to the pathway of melatonin. This pathway may be adversely impacted    even though Tryptophan levels are within a normal range. 5HTP and B6    are advantageously used for improving Cognitive Acuity, Emotional    Balance, Immuno Fitness as discussed below.-   4. (NAC (N-Acetyl-L-Cysteine)+L-Methionine+Selenium)−Strong    antioxidants used to help with energetic efficiency, very useful for    high intensity athletes. These are cofactors for many enzymes in the    tryptophan pathway particularly in the branch related to kynurenine    metabolism. That branch is driven towards compounds which are    neuroprotective by enzymes for which selenium is a cofactor. That    same pathway is also related to the production of compounds that are    involved in mitochondrial function bringing ADP to ATP which is the    principle energetic source for muscular function. Selenium is    recommended when the KYN/TRYP relationship is abnormally high. These    supplements are advantageously used for improving Energetic Fitness,    Emotional Balance, Immuno Fitness as discussed below.-   5. (Melatonin) Helps with sleep. Is the principle sleep hormone. If    problems associated with sleep dysfunction are not resolved first    with diet and exercise intervention, then with probiotic,    tryptophan, 5HTP utilization Melatonin would be recommended to    directly affect sleep function. Melatonin is advantageously used    when individual indicates issues with sleep.-   6. (Vit. B complex)—B vitamins have a direct impact on your energy    levels, brain function, and cell metabolism. Vitamin B complex helps    prevent infections and helps support or promote: cell health.    Vitamin B's are the direct precursors to NAD and NADH, which are    critical factors in the enzyme complex that drive the generation of    energy in mitochondria. They also play an important role in    regulating kynurenine in the tryptophan pathway. Vitamin B is    advantageously used for improving Energetic Fitness, Cognitive    Acuity, Emotional Balance, Gastro Intestinal Fitness, Immuno Fitness    as discussed below.-   7. (Vitamin C+Zinc). Antioxidant and mineral that helps support a    healthy immune system. If there is evidence of oxidative stress    through high 4HBAC, Indoxyl sulfate, and uric acid Vitamin C and    zinc will mediate against the effects of free radical effect of    other biochemical systems. Vitamin C+Zinc is advantageously used for    improving Energetic Fitness, Cognitive Acuity, Gastro Intestinal    Fitness, Immuno Fitness as discussed below.

Numerous variations are possible, depending on the intended audience forthe recommended actions for health improvement 116 as will be discussedbelow.

Periodically, fresh blood samples are taken from an individual, theblood samples processed and metabolites measured as before, revisedrecommendation are provided based on the health/disease state of theindividual, and the process is again repeated periodically at 212.

Further illustrative of the invention is seen in FIG. 4 which a threedimensional graph illustrating measurements of feces samples of elevatedcresol and other phenolics of ALS mouse models treated mouse models andwild type littermates. And, FIGS. 5A and 5B graphs IPA measurements ofwild type and gene positive Huntington's Disease mouse models fromfeces.

FIG. 5A shows one of a series of (two out) tests of the PLS-DA model forassessing the degree to which the foot print of the gut microbiomereflected in the dry weight normalized coordinately bound LCECA patternsof feces allows categorization of young 19 day littermate WT and GPCAG140 mice. Training sets of 8 and validation sets of 2 aresequentially evaluated for all samples. In the example shown both GP andone WT were correctly classified. A similar model for old 90 day WT andGP CAG140 mice is shown in FIG. 5B where three were correctlyclassified. Overall for the young and old categories CCR is 0.93 and0.86 respectively. Thus, we can currently categorize genetic status of amouse by its microbiome foot print about 90% of the time even prior toany symptoms.

Further details of the present disclosure are given in the followingexample of metabolite measurements and recommended supplements:

A) Gastro Intestinal Fitness

Supplements recommended:

-   -   1. Probiotic if tryptophan, tyrosine, ILA and IAA are high, and        if IPA, ILA and IAA are low    -   2. Typtophan if Tryptophan low    -   3. 5HTP and B6 if serotonin is low    -   4. Vitamin C and Zinc if Tyrosine is low and Uric acid is High

B) Immuno Fitness

Supplements might be recommended:

-   -   1. Probiotic if Tryptophan and Indoxyl sulfate are high, or if        IPA and serotonin are low    -   2. Tryptophan if Tryptophan is low    -   3. Vitamin B complex if KYN is high    -   4. 5HTP and B6 if serotonin is low    -   5. NAC+L-Methionine+Selenium if KYN is high    -   6. Vitamin C and Zinc if Tyrosine is low and Uric acid is High

Supplements recommended:

-   -   1. Probiotic if tryptophan is high and Serotonin is low    -   2. Tryptophan if Tryptophan or serotonin is low    -   3. Vitamin B complex if KYN is high    -   4. 5HTP and B6 if serotonin is low    -   5. Vitamin C and Zinc if KYN is high

C) Cognitive Acuity

Supplements recommended

-   -   1. Probiotic if tryptophan and tyrosine are high and IPA and        Serotonin are low    -   2. Vitamin C and Zinc if Tyrosine is high    -   3. Tryptophan if Tryptophan or serotonin is low    -   4. Vitamin B complex if Kyn is high    -   5. 5HTP and B6 if serotonin is low

Supplements that might be recommended

-   -   1. Probiotic if tryptophan and tyrosine are high    -   2. Vitamin C and Zinc if xanthine, tyrosine, or uric acid are        high    -   3. Tryptophan if Tryptophan is low    -   4. Vitamin B complex if KYN is high    -   5. NAC plus LMethionine and Selenium if Xanthine is high

Following are blood test results, before and after, if variousindividuals based on administration of various supplements.

Metabolite Unit 3MX ng/ml IAA ng/ml ILA ng/ml IPA ng/ml IDS ng/ml Normal<500 30-300 50-500 >55 <400 Indiv. 1 Before¹ 86.09 141.25 129.45 203.11315.65 After 6.21 92.5 91.16 117.25 274.54 Indiv. 2 Before² 54.84 145.08123.50 51.76 409.55 After 32.68 167.81 125.67 100.16 357.57 Indiv. 3Before³ 36.07 228.97 123.35 129.94 497.11 After 5.17 169.51 91.2 537.27157.68 Indiv. 4 Before ⁴ 33.2 154.47 113.4 142.41 233.62 After 69.04121.55 93.74 85.22 333.65 Indiv. 5 Before⁵ 25.38 138.72 87.64 44.57146.84 After 104.88 87.23 117.54 61.66 236.79 Indiv. 6 Before⁶21.44986478 139.0087441 64.89523 37.2547 215.4566 After 17.61423478205.2114178 96.49815 57.22367 227.9377 Indiv. 7 Before⁷ 80.95 131.70109.46 52.552 489.62 After 171.992 156.698 65.428 71.62 332.48 Indiv. 8Before⁸ 83.23 118.45 70.11 45.62 228.80 After 64.93 138.45 67.69 54.29361.55 Indiv. 9 Before ⁹ 110.19 97.31 32.73 87.41 272.04 After 107.18127.90 44.35 165.35 109.00 Indiv. 10 Before¹⁰ 19.26672 132.6309 57.7386612.73788 92.22044 After 132.9447 123.946 60.5723 24.7631 168.1507Metabolite KYN serotonin tryptophan Tyrosine uric acid xanthine Unitng/ml ng/ml μg/ml μg/ml μg/ml ng/ml Normal 75-350 15-120 4.1-9.54.5-20.5 15-42 30-190 Indiv. 1 Before 356.69 82.68 7.25 9.69 21.05248.99 After 233.29 82.45 6.04 7.46 19.46 156.25 Indiv. 2 Before 186.6661.20 7.22 10.94 30.12 233.86 After 209.94 75.19 7.27 8.91 30.99 130.94Indiv. 3 Before 218.01 68.08 7.01 8.59 27.38 221.81 After 206.62 70.276.45 6.52 28.89 184.37 Indiv. 4 Before 301.62 48.23 8.47 11.45 24.61222.94 After 285.79 49.07 6.75 8.14 26.21 165.99 Indiv. 5 Before 256.4128.09 6.44 9.16 15.9 330.96 After 202.89 42.74 8.01 14.16 19.9 144.6Indiv. 6 Before 163.8883 17.41096 6.922951 8.830608 20.34835 140.299After 169.4513 21.07541 6.619294 6.584736 16.86458 148.6497 Indiv. 7Before 671.44 196.33 7.16 11.65 28.71 188.35 After 135.92 30.354 5.0286.65 13.19 80.456 Indiv. 8 Before 280.60 7.63 4.26 7.75 21.59 96.20After 244.80 47.30 4.50 7.23 20.29 83.65 Indiv. 9 Before 161.91 29.095.07 7.94 13.47 86.72 After 196.80 39.45 5.74 9.68 14.92 98.03 Indiv. 10Before 156.2026 47.10654 5.63586 7.49808 27.86688 103.0789 After 167.96852.4378 6.1306 10.0195 29.6989 182.7638 ¹Probiotic, diet modificationsand VitC, zinc, CoQ10, B complex ²Probiotic, diet modifications andVitC, zinc, CoQ10 ³Probiotic, and VitC, zinc, CoQ10 ⁴ VitC, zinc, CoQ10⁵Probiotic, and VitC, zinc, CoQ10 ⁶probiotic ⁷Probiotic, vit B complex ⁸Probiotic, 5HTP ⁹ probiotic ¹⁰probiotic

Individual 1 was given a specially designed 17 strain Probiotic, haddiet modifications suggested and supplements of Vitamin C, zinc, CoQ10,B complex were added. This helped to lower Indoxyl Sulfate, Kynurenine,Xanthine

Individual 2 was given a specially designed 17 strain Probiotic, haddiet modifications suggested and supplements of Vitamin C, zinc, CoQ10.Lowered Xanthine and IDS and raised IPA.

Individual 3 was given a specially designed 17 strain Probiotic andsupplements of Vitamin C, zinc, CoQ10. Lowered Indoxyl sulfate andXanthine

Individual 4 was given supplements of Vitamin C, zinc, CoQ10. LoweredXanthine.

Individual 5 was given a specially designed 17 strain Probiotic andsupplements of Vitamin C, zinc, CoQ10. Lowered xanthine, raised IPA andserotonin

Individual 6 was given a specially designed 17 strain Probiotic. Beganto raise levels of serotonin and IPA

Individual 7 was given a specially designed 17 strain Probiotic andsupplemented with vitamin B complex. Lowered Kynurenine, serotonin andIndoxyl sulfate and raised IPA.

Individual 8 was given a specially designed 17 strain Probiotic andsupplement 5HTP. Raised serotonin and IPA.

Individual 9 was given a specially designed 17 strain Probiotic. LoweredIndoxyl sulfate, raise IPA and ILA.

Individual 10 was given a specially designed 17 strain Probiotic. RaisedIPA

Various changes may be made without departing from the spirit and scopeof the disclosure. All such modifications and variations are intended tobe included herein within the scope of this disclosure and the presentdisclosure and protected by the following claims.

What is claimed is:
 1. A method for improving the gut health, emotionalbalance, cognitive acuity, energetic efficiency and immuno fitness of anindividual in need of improvement, the method consisting of the steps insequence of: (1) collecting fluid samples from said individual; (2)analyzing said fluid samples for concentration of the followingmicrobiome modified compounds in the fluid samples: indole propionicacid (IPA), indole-3-lactic acid (ILA), indole-3-acetic acid (IAA),Tryptophan (TRP), Serotonin (SER), Kynurenine (KYN), Total IndoxylSulfate (IDS), Tyrosine (TYR), Xanthine (XAN) and Uric Acid (UA); (3)comparing the concentration of said microbiome modified compounds tostandards concentrations; (4) providing for oral administration to saidindividual one or more supplements selected from the group consisting ofa prebiotic, a probiotic, a vitamin B complex, and CoQ10 protocol toraise or reduce the concentration, as the case may be, of components ofthe gut microbiome of said individual that effect the one or moremicrobiome modified compounds that have measured concentrations thatfall outside of the standards concentrations; and (5) repeating steps(1) to (4) to bring the measured concentrations to within the standardsconcentrations.
 2. The method according to claim 1, wherein said liquidsamples comprise blood.
 3. The method according to claim 1, wherein saidliquid samples comprise urine.
 4. The method according to claim 1,wherein said liquid samples comprise tears.
 5. The method according toclaim 1, wherein two or more supplements selected from the groupconsisting of a prebiotic, a probiotic, a vitamin B complex, and CoQ10are administered.
 6. The method according to claim 1, wherein saidstandards concentrations are created by collecting and analyzing fluidsamples from a plurality of different individuals.
 7. A method forimproving the gut health, emotional balance, cognitive acuity, energeticefficiency and immuno fitness of an individual in need of improvement,the method consisting of the steps in sequence of: (1) collecting fluidsamples from said individual; (2) analyzing said fluid samples forconcentration of the following microbiome modified compounds in thefluid samples: indole propionic acid (IPA), indole-3-lactic acid (ILA),indole-3-acetic acid (IAA), Tryptophan (TRP), Kynurenine (KYN), TotalIndoxyl Sulfate (IDS), Tyrosine (TYR), Xanthine (XAN), 3-Methylxanthine(3MXAN) and Uric Acid (UA); (3) comparing the concentration of saidmicrobiome modified compounds to standards concentrations; (4) providingfor oral administration to said individual one or more supplementsselected from the group consisting of a prebiotic, a probiotic, avitamin B complex, and CoQ10 protocol to raise or reduce theconcentration, as the case may be, of components of the gut microbiomeof said individual that effect the one or more microbiome modifiedcompounds that have measured concentrations that fall outside of thestandards concentrations; and (5) repeating steps (1) to (4) to bringthe measured concentrations to within the standards concentrations. 8.The method according to claim 7, wherein said liquid samples compriseblood.
 9. The method according to claim 7, wherein said liquid samplescomprise urine.
 10. The method according to claim 7, wherein said liquidsamples comprise tears.
 11. The method according to claim 7, wherein twoor more supplements selected from the group consisting of a prebiotic, aprobiotic, a vitamin B complex, and CoQ10 are administered.
 12. Themethod according to claim 7, wherein said standards concentrations arecreated by collecting and analyzing fluid samples from a plurality ofdifferent individuals.
 13. A method for improving the gut health,emotional balance, cognitive acuity, energetic efficiency and immunofitness of an individual in need of improvement, the method consistingof the steps in sequence of: (1) collecting fluid samples from saidindividual; (2) analyzing said fluid samples for concentration of thefollowing microbiome modified compounds in the fluid samples: indolepropionic acid (IPA), indole-3-lactic acid (ILA), indole-3-acetic acid(IAA), Tryptophan (TRP), Serotonin (SER), Kynurenine (KYN), TotalIndoxyl Sulfate (IDS), Tyrosine (TYR), 3-Methylxanthine (3MXAN) and UricAcid (UA); (3) comparing the concentration of said microbiome modifiedcompounds to standards concentrations; (4) providing for oraladministration to said individual one or more supplements selected fromthe group consisting of a prebiotic, a probiotic, a vitamin B complex,and CoQ10 protocol to raise or reduce the concentration, as the case maybe, of components of the gut microbiome of said individual that effectthe one or more microbiome modified compounds that have measuredconcentrations that fall outside of the standards concentrations; and(5) repeating steps (1) to (4) to bring the measured concentrations towithin the standards concentrations.
 14. The method according to claim13, wherein said liquid samples comprise blood.
 15. The method accordingto claim 13, wherein said liquid samples comprise urine.
 16. The methodaccording to claim 13, wherein said liquid samples comprise tears. 17.The method according to claim 13, wherein two or more supplementsselected from the group consisting of a prebiotic, a probiotic, avitamin B complex, and CoQ10 are administered.
 18. The method accordingto claim 13, wherein said standards concentrations are created bycollecting and analyzing fluid samples from a plurality of differentindividuals.